EasySep™人泛b细胞富集试剂盒

未接触人泛b细胞的免疫磁阴性分离

率先评论此产品

产品号 #（选择产品）

产品号 #19554_C

未接触人泛b细胞的免疫磁阴性分离

产品优势

快速，易于使用和无列
纯度高达99%
分离的细胞不受影响

产品组分包括

EasySep™人泛b细胞富集试剂盒（目录#19554）

EasySep™人Pan-B细胞富集试剂盒，1ml
EasySep™磁性颗粒，2 × 1ml

RoboSep™人泛b细胞富集试剂盒（目录#19554RF）

EasySep™人Pan-B细胞富集试剂盒，1ml
EasySep™磁性颗粒，2 × 1ml
RoboSep™缓冲器（目录#20104）
RoboSep™过滤器提示(目录#20125)

跳到结尾的图片库

跳转到图像库的开头

概述

使用EasySep™人Pan-B细胞富集试剂盒，通过免疫磁阴性选择，从新鲜或先前冷冻的人外周血单个核细胞（PBMCs）或裂解的白细胞分离样品中轻松高效地分离高度纯化的人B细胞，包括浆细胞。EasySep™在已发表的研究中广泛使用了20多年，它结合了单克隆抗体的特异性和无柱磁系统的简单性。

在这个EasySep™阴性选择程序中，用抗体复合物和磁性颗粒标记不需要的细胞。表达以下标记的不需要的细胞被靶向去除：CD2， CD3, CD14, CD16, CD36, CD42b, CD56, CD66b， CD123和glyA。然后使用EasySep™磁铁将磁性标记的细胞与未接触的所需B细胞分离，并将所需细胞倒入或移液到新管中。磁性细胞分离后，所需的B细胞可用于流式细胞术、培养或DNA/RNA提取等下游应用。

对于仅从正常样本中分离cd43阴性B细胞，我们建议使用EasySep™人B细胞富集试剂盒(目录# 19054)。

对于B细胞白血病或淋巴瘤患者外周血或其他组织中B细胞的富集，或B细胞可能表达CD43、CD36和/或CD123的其他疾病状态，我们推荐使用EasySep™人B细胞富集试剂盒II (CD43缺失(目录# 17963)。

了解更多关于免疫磁性的知识EasySep™技术工作原理或如何完全自动化免疫磁细胞分离RoboSep™。探索额外的产品针对您的工作流程进行了优化，包括培养基、补充剂、抗体等。

Data Figures

Figure 1. Typical EasySep™ Human Pan-B Cell Enrichment Profile

Starting with nucleated cells, the pan-B cell [Lineage (CD4, CD8, CD14, CD16, CD56) negative, CD19+ and CD19-CD43+] content of the enriched fraction typically ranges from 90 - 99%.

Figure 2. Expansion and Maturation of Human B Cells with ImmunoCult™ Human B Cell Expansion Kit

B cells isolated from human peripheral blood mononuclear cells (PBMCs) using EasySep™ Human Pan-B Cell Enrichment Kit (Catalog #19554) were seeded at 1 x 10⁵ cells/well in 24-well tissue culture plates with ImmunoCult™-ACF Human B Cell Expansion Supplement and ImmunoCult™-XF B Cell Base Medium, included in the ImmunoCult™ Human B Cell Expansion Kit (Catalog #100-0645). The cells were passaged every 3 - 4 days.

(A) Fold expansion of viable cells is shown for n = 12 donors, with bars representing the mean and 95% confidence level (range 38- to 1190-fold at Day 14 ± 1 day).

(B) Expression of CD138 and CD20 was analyzed by flow cytometry at each timepoint (data represent % positive viable cells; mean ± 1 SD). The observed changes indicate maturation of B cells to plasma cells/blasts.

Figure 3. Light Microscopy Image of Cultured Human B Cells

B cells isolated from human PBMCs using EasySep™ Human Pan-B Cell Enrichment Kit (Catalog #19554) were seeded at 1 x 10⁵ cells/well in a 24-well tissue culture plate and cultured with the ImmunoCult™ Human B Cell Expansion Kit (Catalog #100-0646). The cells were passaged on Day 4 after seeding and imaged at 40X magnification on Day 6.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Frequently Asked Questions

Can EasySep™ be used for either positive or negative selection?

Yes. The EasySep™ kits use either a negative selection approach by targeting and removing unwanted cells or a positive selection approach targeting desired cells. Depletion kits are also available for the removal of cells with a specific undesired marker (e.g. GlyA).

How does the separation work?

Magnetic particles are crosslinked to cells using Tetrameric Antibody Complexes (TAC). When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a separate fraction.

Which columns do I use?

The EasySep™ procedure is column-free. That's right - no columns!

How can I analyze the purity of my enriched sample?

The Product Information Sheet provided with each EasySep™ kit contains detailed staining information.

Can EasySep™ separations be automated?

Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.

Can EasySep™ be used to isolate rare cells?

Yes. We recommend a cell concentration of 2x10⁸ cells/mL and a minimum working volume of 100 µL. Samples containing 2x10⁷ cells or fewer should be suspended in 100 µL of buffer.

Are the EasySep™ magnetic particles FACS-compatible?

Yes, the EasySep™ particles are flow cytometry-compatible, as they are very uniform in size and about 5000X smaller than other commercially available magnetic beads used with column-free systems.

Can the EasySep™ magnetic particles be removed after enrichment?

No, but due to the small size of these particles, they will not interfere with downstream applications.

How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?

Hundreds of publications have used cells selected with EasySep™ positive selection kits for functional studies. Our in-house experiments also confirm that selected cells are not functionally altered by the EasySep™ magnetic particles.

If particle binding is a key concern, we offer two options for negative selection. The EasySep™ negative selection kits can isolate untouched cells with comparable purities, while RosetteSep™ can isolate untouched cells directly from whole blood without using particles or magnets.

Can I alter the separation time in the magnet?

Yes; however, this may impact the kit's performance. The provided EasySep™ protocols have already been optimized to balance purity, recovery and time spent on the isolation.

For positive selection, can I perform more than 3 separations to increase purity?

Yes, the purity of targeted cells will increase with additional rounds of separations; however, cell recovery will decrease.

Which cell separation kits are compatible with the "Easy 50" EASYSEP™ magnet?

At present, the "Easy 50" EasySep™ magnet is only compatible with EasySep™ kits for human cell separation.

T Cells: 19051 (T Cells), 19052 (CD4 T cells), 19157 (Memory CD4 T Cells), 19053 (CD8 T Cells), 19159 (Memory CD8 T Cells - please contact Tech Support)

B Cells: 19054 (B Cells), 19254 (Naïve B cells)

Other Cell Types: 19055 (NK Cells), 19058 (Monocytes without CD16 depletion), 19059 (Monocytes), 19062 (Plasmacytoid DCs), 19251 (pan-DCs)

For HLA Analysis: 19951HLA (T Cells from whole blood), 19954HLA (B Cells from whole blood), 19961HLA (Total lymphocytes from whole blood)

Publications (5)

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires. E. E. Ford et al. Journal of immunology (Baltimore, Md. : 1950) 2023 may

Abstract

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

View publication

Germinal centre-driven maturation of B cell response to mRNA vaccination. W. Kim et al. Nature 2022 apr

Abstract

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.

View publication

B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection. T. Pattarabanjird et al. Circulation research 2022 apr

Abstract

BACKGROUND B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. METHODS Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS Through RNA sequencing, P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-$\kappa$B (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.

View publication

View All Publications

更多信息

更多信息
Species	Human
Magnet Compatibility	• EasySep™ Magnet (Catalog #18000) • “The Big Easy” EasySep™ Magnet (Catalog #18001) • Easy 50 EasySep™ Magnet (Catalog #18002) • RoboSep™-S (Catalog #21000)
Sample Source	Leukapheresis, PBMC
Selection Method	Negative

EasySep™人泛b细胞富集试剂盒

产品号 #（选择产品）

产品号 #19554_C

产品优势

产品组分包括

概述

Data Figures

Protocols and Documentation

Applications

Resources and Publications

Request Pricing

更多信息

Scientists Helping Scientists™

EasySep™人泛b细胞富集试剂盒

产品号 #（选择产品）

产品号 #19554_C

产品优势

产品组分包括

概述

Data Figures

Protocols and Documentation

Applications

Resources and Publications

Educational Materials (9)

Frequently Asked Questions

Can EasySep™ be used for either positive or negative selection?

How does the separation work?

Which columns do I use?

How can I analyze the purity of my enriched sample?

Can EasySep™ separations be automated?

Can EasySep™ be used to isolate rare cells?

Are the EasySep™ magnetic particles FACS-compatible?

Can the EasySep™ magnetic particles be removed after enrichment?

How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?

Can I alter the separation time in the magnet?

For positive selection, can I perform more than 3 separations to increase purity?

Which cell separation kits are compatible with the "Easy 50" EASYSEP™ magnet?

Publications (5)

Abstract

Abstract

Abstract

Request Pricing

更多信息

欢迎您关注STEMCELL Technologies的微信公众平台